SECRET AGENT O-GlcNAc Modifies GIGANTEA: Methods for Mapping of O-Linked β-N-Acetylglucosamine Modification Sites Using Mass Spectrometry

Abstract

Arabidopsis thaliana has two glycosyl transferases, SPINDLY (SPY) and SECRET AGENT (SEC), that modify nuclear and cytosolic protein with O-linked fucose and N-acetylglucosamine (GlcNAc), respectively. SPY interacts physically and genetically with GIGANTEA (GI). Previously, we reported that SEC substrates are O-GlcNAc modified when they are co-expressed in E. coli. By analyzing overlapping sub-fragments of GI, we found a region that was modified by SEC. Mutational mapping of the modified region was then performed. Modification was undetectable when threonine 829 was mutated to alanine (T829A) while the T834A and T837A mutations reduced modification suggesting that T829 was the primary or only modification site. Mapping using several enrichment and mass spectrometry methods all detected only modification of T829.